There are five subtypes of dopamine receptors, classified into two primary categories, D1-like and D2-like. The D1-like family consists of the D1 and D5 dopamine receptors, whereas, the D2-like family consists of the D2, D3, and D4 dopamine receptors. The D1 dopamine receptor (D1R) is associated with a number of neuropsychiatric disorders, such as Parkinson's disease and cognitive decline associated with schizophrenia and Alzheimer's disease. Positive allosteric modulators (PAMs) of the D1R have emerged as a drug development strategy as D1R orthosteric agonists have clinical liabilities curtailing their use. We recently discovered two D1R PAMs via a high-throughput screen, MLS1082 and MLS6585. These two PAMs act similarly, potentiating agonist-stimulated G-protein and beta-arrestin-mediated signaling and increasing dopamine affinity for the D1R. Neither compound has agonist activity in the absence of dopamine. MLS1082 and MLS6585 are structurally distinct chemical series and we found that they act at two separate binding sites. MLS1082 shares a similar binding site with two other known D1 PAMs, Compound B and DETQ, which involves the R130 amino acid in the second intracellular loop of the D1R. MLS6585 displays additive activity with MLS1082, Compound B, and DETQ, and its activity is not affected by an R130Q point mutation, strongly suggesting that MLS6585 acts at a separate binding site than these other D1R PAMs. We used several analogs of both MLS1082 and MLS6585 to improve on the PAM activity of the parent compounds. Many analogs of both MLS1082 and MLS6585 retained PAM activity, validating these drug scaffolds as D1R modulators. The analogs of one series displayed additive activity with the opposite series, suggesting that the analogs are binding to similar sites as their parent compounds. A small number of seemingly inactive analogs appear to act as silent allosteric modulators (SAMs), in that they blocked the activity of the parent PAM compound, presumably by competing for the same binding site. Further analog studies are underway to better understand the chemical SAR needed for D1R allosteric modulation as well as the differences between the two D1R PAM binding sites. Despite its clinical importance in the treatment of a number of neuropsychiatric disorders, such as Parkinson's disease and schizophrenia, there are few compounds that are highly selective for the D2 dopamine receptor (D2R). Most compounds with activity at the D2R also exhibit significant affinity for the D3 or D4 receptors (D3R or D4R), or other G protein-coupled receptors (GPCRs). In FY19, we continued optimizing a highly selective antagonist for the D2R that we previously identified via a high-throughput screen - ML321. In a functional profiling screen of 168 different GPCRs, ML321 shows relatively little activity beyond inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. PET imaging studies in non-human primates demonstrate that ML321 can penetrate the CNS and occupy the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that that ML321 can selectivity antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus demonstrating good in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 can attenuate both PCP- and amphetamine-induced locomotor activity and pre-pulse inhibition (PPI) in a dose-dependent manner. ML321 also attenuates the hyperactivity seen in DA transporter (DAT) knockout mice. Importantly, using doses that are maximally effective in the locomotor and PPI studies, ML321 promotes little catalepsy compared with the non-selective antipsychotic haloperidol. These latter observations suggest that ML321 may produce fewer extrapyramidal motor side-effects (EPS), a common problem with FDA-approved antipsychotics. Overall, these results suggest that the ML321 scaffold can serve as a lead compound for the development of an improved therapeutic with greatly reduced side-effects for treating schizophrenia and other psychotic syndromes. Signaling bias is the propensity for some agonists to preferentially stimulate G protein-coupled receptor (GPCR) signaling through one intracellular pathway versus another. While GPCR agonists have been described that selectively activate G proteins or beta-arrestins, the molecular mechanisms underlying biased signaling are not well understood. We recently identified a G protein-biased agonist of the D2 dopamine receptor (D2R) that exhibits impaired beta-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. We have shown that residue F189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a micro-switch for regulating receptor interactions with beta-arrestin. As this residue is relatively conserved among class A GPCRs, we constructed analogous mutations within other GPCRs and found that these alterations similarly impaired beta-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active state structure of the beta2-adrenergic receptor (beta2R), to build beta2R-WT and beta2R-Y1995.38A models in complex with the full beta2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in beta2R-Y1995.38A that propagate from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in beta2R-Y1995.38A, which is predicted to affect its interactions with beta-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions resulting in biased signaling. Due to the limited distribution of the D3R in limbic regions of the brain, D3R-selective antagonists may be useful as therapeutics for substance use disorders as they could attenuate drug craving symptoms without the motor side effects frequently incurred by D2R-preferring antagonists. However, high sequence homology shared by the D2R and D3R within their orthosteric binding sites has made the discovery of subtype-selective compounds difficult. In an effort to discover highly selective allosteric antagonists for the D3R, our lab employed a high-throughput screen of the NIH Molecular Libraries Program 400,000+ small molecule library. The library was initially screened using a D3R-mediated beta-arrestin recruitment assay. We advanced one compound for medicinal chemistry efforts after further triaging of the hits with confirmation and counter-screens. This compound, MLS6357, was found to be D3R versus D2R selective in several functional outputs including beta-arrestin recruitment and G-protein activation. Furthermore, Schild-type functional assays indicate that this compound acts in a purely non-competitive manner at the D3R. In addition, binding and functional screens of closely related GPCRs indicate that this compound has limited cross-reactivity with other receptors. 30 analogs of this scaffold have been tested for activity and D3R selectivity, yielding SAR information for further refinement of the scaffold, and one analog was identified that was 15-fold more potent than the parent. Further medicinal chemistry and determination of the binding site of this scaffold is ongoing. This pharmacophore may prove useful as a pharmacological probe or therapeutic lead for D3R-related pathophysiology.